The long-term objective of this proposal is to elucidate the biosynthetic and regulatory mechanisms that govern inorganic nitrogen metabolism and signaling. Our previous work has focused on nitrate, which serves as a nutrient and signal for plant growth. This proposal focuses on the synthesis of nitric oxide (NO) and how it serves as a signal in growth and development. NO is an unusual signal in that it is a reactive gas, yet it regulates a large number of cellular and physiological processes including respiration, blood flow and neural development in animals and germination, defence and flowering in plants. In animals, NO is made from arginine by nitric oxide synthase (NOS) enzymes. In plants, NO can be made by the reduction of nitrite by the enzyme nitrate reductase. Plants also have an arginine-dependent activity, but no enzyme similar to animal NOS proteins has been identified. We have discovered a gene (AtNOS1) that is needed for arginine-dependent NO synthesis in Arabidopsis. This protein has NOS activity in vitro but is not related to animal NOS in sequence and thus may be a novel NOS. AtNOS1 is involved in hormonal signaling, is located in the mitochondria, and is needed for arginine-stimulated NO production by isolated mitochondria. Such synthesis is important as NO regulates mitochondrial functions including respiration, caspase-mediated cell death, and biogenesis, which affects obesity and longevity. There are 2 sources of mitochondrial NO in animals: eNOS and mtNOS, the identity of the latter being still in question. Because mammalian mitochondria have proteins similar in sequence to AtNOS1, it is possible that AtNOS1-like proteins are or contribute to mtNOS. Our proposal is to investigate the biochemical activity and functions of AtNOS1 and its sister genes AtNOS2 and AtNOS3. Specifically, we will determine the biochemical mechanism for NO production by AtNOS1, determine the role of AtNOS1 in mitochondrial function, elucidate the role of NO synthesis and AtNOS1 function in hormonal signaling, and elucidate the functions and biochemical mechanisms of AtNOS2 and AtNOS3. These studies should shed light on not only the mechanism for NO synthesis used in signaling in plants but also the production of NO in mammalian mitochondria. They also may provide a new mechanism for NO synthesis conserved in plants and animals. [unreadable] [unreadable] [unreadable]